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REGULAR ARTICLES |
The Fred Hutchinson Cancer Research Center and the University of Washington, Seattle, Washington.
Correspondence to Dr. Richard A. Zager, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Room D2-190, Seattle, WA 98109. Phone: 206-667-6549; Fax: 206-667-5424; E-mail: dzager{at}fhcrc.org
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Each of these processes has potential implications for both the development of, and recovery from, acute renal failure (ARF). Therefore, several recent studies have focused on whether ceramide accumulation is a consequence of acute ischemic/hypoxic and toxic proximal tubular cell attack. Indeed, this appears to be the case, because within several minutes of inducing acute tubular damage, approximately 50 to 200% ceramide increments result (13,14,15,16,17). These elevations persist for at least 24 h post-renal injury, indicating that they do not simply represent a transient biologic event. Multiple biochemical pathways can contribute to this ceramide accumulation, including de novo synthesis (17), decreased ceramide catabolism (13), and SMase-mediated SM hydrolysis. The latter may then potentially contribute to injury-induced decrements in proximal tubular brush border membrane SM content (18,19,20).
These types of observations have stimulated investigation of the "SMase-ceramide pathway" as a determinant of acute cell injury and repair. The general experimental approach has been to subject cultured cells to either "chemical hypoxic" or toxic injury in the presence or absence of exogenous ceramide treatment. Depending on the type of injury model studied, both injury potentiation (e.g., hypoxia) (16,17) and attenuation (e.g., iron-mediated oxidant stress; arachidonic acid toxicity (13,16) have been observed. While these findings underscore that ceramide can, indeed, influence tubular injury responses, they cannot be readily extrapolated to SMase-mediated ceramide generation for a number of important reasons. First, exogenous ceramide treatment tends to increase, rather than decrease, cellular SM content because provision of ceramide provides a substrate for de novo SM synthesis. This is the opposite situation to SMase-mediated ceramide generation, in which SM decrements, rather than increments, result. Second, commercially available long chain (physiologic) ceramides gain minimal, if any, intracellular access. Hence, when they are added to cultured cells, they cannot recapitulate the same membrane orientation that results from membrane SM hydrolysis. Third, the term "ceramide" refers to a heterogeneous family of compounds, consisting of sphingosine joined by amide linkage to any one of a number of fatty acids. For example, we have recently observed that after ischemic or toxic renal injury in the mouse, at least nine distinct ceramides accumulate, possibly in a disease-specific manner (15). Because most physiologic ceramides are not commercially available, one cannot simply add exogenous ceramides to re-create these ceramide accumulation patterns.
In view of these considerations, the present study has sought to identify the impact of SMase activity, and not simply exogenous ceramide treatment, on the expression of evolving renal tubular cell damage. To this end, cultured human proximal tubular (HK-2) cells were exposed to exogenous SMase, producing ceramide generation in the setting of SM hydrolysis. The impact of these changes on the expression of superimposed acute toxic and "chemical hypoxic" cell injury was then assessed. In addition, SMase's effects on cellular proliferation, both under basal conditions and after tubular cell injury, were assessed. The results of these studies form the basis of this report.
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Materials and Methods |
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Effects of SMase on ATP Depletion/Ca2+ Ionophore-Induced
Injury
HK-2 cells were grown within cluster plates under routine culture
conditions for 24 to 48 h. Then, each 24-well plate was divided into six
groups, 4 wells per group, as follows: (1) continued control culture
conditions; (2) addition of 1 U/ml bacterial sphingomyelinase (SMase;
from Staphylococcus aureus, catalogue no. SE 108, Biolmol; Plymouth
Meeting, PA); (3) a combined challenge of ATP depletion (7.5 µM
antimycin A/20 mM 2-deoxyglucose) + cytosolic Ca2+ overload (10
µM Ca2+ ionophore A23187), as described previously
(22); and (4) the ATP
depletion/Ca2+ ionophore challenge ("CAD" =
Ca2+ ionophore + Antimycin +
Deoxyglucose) conducted in the presence of 1 U/ml SMase. Because the
SMase is provided in a glycerol carrier (final concentration of 0.125% when
added to the cells), this same glycerol concentration was added to those cell
wells that were paired to SMase treatment groups. (These same conditions were
used with all subsequent SMase experiments described below.) After an
overnight incubation (approximately 14 h), the extent of cell injury was
assessed by percentage of lactate dehydrogenase (LDH) release
(13). Each of these treatments
was conducted on at least 4 wells of cells on four separate occasions.
(Note: The 1 U/ml dose of SMase was selected for these experiments
because pilot studies demonstrated that it caused both SM reductions and
ceramide increases without exerting an independent cytotoxic effect. See
Results.)
Effect of SMase on Fe-Mediated Oxidative Stress
HK-2 cells were cultured in 24-well cluster plates for 24 to 48 h and then
wells within each plate were treated as follows: (1) continued
control incubation; (2) treatment with 1 U/ml SMase; (3)
Fe-mediated oxidative stress (7.5 µM ferrous ammonium sulfate, complexed to
7.5 µM 8-hydroxyquinoline [HQ], which facilitates Fe's intracellular
access)
(13,23);
and (4) combined simultaneous treatment with SMase + FeHQ. After
completing an overnight 14-h incubation, the extent of lethal cell injury was
assessed by calculating percentage of LDH release. Each of these treatments
was conducted on at least 4 wells of cells on four separate occasions.
SMase Effects on the Expression of Arachidonic Acid Cytotoxicity
Previous studies have demonstrated that exogenous ceramide addition to
cells can antagonize arachidonic acid's (C20:4) cytolytic effects
(13,16).
The following experiment explored whether a blunting of C20:4 toxicity can
also be observed under conditions of SMase-mediated ceramide generation. To
this end, HK-2 cells within 24-well cluster plates were divided into four
equal groups (6 wells each) as follows: (1) control incubation;
(2) incubation with 1 U/ml of SMase; (3) C20:4 addition (25
µM; in ethanol; final concentration, 0.125%); and (4) combined
SMase + C20:4 treatment. After completing a 14-h exposure, cell injury was
gauged by percentage of LDH release. This experiment was repeated on four
separate occasions, n = 6 wells each.
SMase Effects on HK-2 Cell Phospholipid, Cholesterol, and Ceramide
Content
The following experiments ascertained the impact of SMase treatment on HK-2
cell SM and ceramide concentrations. In addition, its effects on other major
membrane phospholipid classes and total cholesterol levels were assessed.
Cholesterol levels were determined because: (1) cholesterol can
independently influence tubular cell vulnerability to injury
(24); and (2) SMase
has been reported to decrease cell cholesterol content in selected cell lines
(4,25).
Phospholipids. HK-2 cells were grown in eight T75 Costar culture flasks. After reaching near confluence (3 d), the cells were treated for 14 h with either 1 U/ml SMase or with SMase vehicle (n = 4 each). The cells were harvested from the flasks by detachment with a rubber policeman followed by centrifugation, as described previously (13). The recovered cells underwent chloroform:methanol extraction, and the lipids were analyzed by two-dimensional thin-layer chromatography (TLC) (26). Specifically, the recovered lipids were analyzed for SM, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) content. The amount of each phospholipid was quantified by the amount of recovered phosphate in each phospholipid spot recovered from the TLC plate (26). The values were reported as the percentage to which each individual phospholipid contributed to the sum of the total quantified phospholipid mass (sum of SM + PC + PE + PI + PS).
Cholesterol Content. Six T75 flasks were seeded with HK-2 cells, and when near confluence, they were subjected to either continued control incubation or to SMase treatment, as noted above (n = 3 each). After completing a 14-h SMase challenge, the cells were recovered from each of the flasks as noted above, and then they were assayed for total cholesterol content (cholesterol assay kit; No. 352; Sigma Chemical Co., St. Louis, MO), as described previously (24). In brief, this assay subjects total cholesterol to treatment with cholesterol esterase (to deacylate cholesterol esters) plus cholesterol oxidase. The latter then converts cholesterol to cholesterol-4-en-3-one + H2O2, the latter being quantified by a colorimetric technique. Cholesterol concentrations were expressed as nmoles/nmole phospholipid phosphate in the recovered HK-2 cell extracts.
Ceramide Content. Six T75 flasks were treated as above, three with and three without SMase for 14 h, and then they were subjected to lipid extraction. The samples were analyzed for individual ceramides, as determined by their specific constituent fatty acid (C16:0, C16:1, C22:0, C22:1, C24:0, C24:1, C24:2, C24:3). These analyses were conducted by a recently described liquid chromatography/mass spectrometric technique developed in this laboratory (15). Individual and total ceramides were expressed as pmoles ceramide/nmole phospholipid phosphate in the cell extracts.
Exogenous SM Treatment: Impact on ATP Depletion and Fe-Mediated Cell
Injury
To assess whether SMase might affect cell injury via changes in SM, and not
simply by generating ceramides, the effect of exogenous SM on cell injury
responses was assessed. Three plates of HK-2 cells were incubated with either
exogenous SM (500 µM; from bovine brain; S7004; Sigma Chemical Co.) or with
the SM carrier (ethanol; final concentration, 0.5%) within 1 h of seeding them
into 24-well cluster plates. After an 8-h exposure, the cells were either left
under these same conditions or they were challenged with the ATP
depletion/Ca2+ ionophore ("CAD") injury protocol, as
described above. The extent of cell injury was assessed 14 h later by
determining percentage of LDH release.
To further determine SM's impact on cell injury, the above protocol was repeated, substituting FeHQ for the CAD challenge. Once again, cell injury was determined 14 h later by LDH release.
Renal Cortical Vesicle Experiments: SM Effects on Membrane
Injury
The following experiments were undertaken to directly assess SM's impact on
membrane damage, independent of integrated intact cell injury responses (such
as metabolic conversions of test reagents).
SM Addition Experiments. Renal cortices were collected from eight mice, and the tissues were subjected to lipid extraction, as described previously (24,26). The total amount of recovered phospholipid was determined by phosphate assay (27). The samples were dissolved at a concentration of 500 nmol phospholipid/100 µl chloroform:methanol (2:1). To this extract was added differing amounts of exogenous SM (from bovine brain in ethanol; see above), creating 5, 10, and 20% additions (by phosphate content). The samples were redried under N2, and then 800 µl of Hanks' balanced salt solution (with Ca and Mg) were added, followed by sonication for 30 min, forming vesicles.
Phospholipase A2-Mediated Membrane Damage. Vesicles with 0, 5, 10, and 20% SM supplementation were incubated with Naja phospholipase A2 (PLA2; 0.05 U/ml; No. P 7778; Sigma Chemical Co.). After completing the incubations, the samples were reextracted in chloroform:methanol and subjected to two-dimensional TLC (26). PLA2-mediated phospholipid breakdown was gauged by quantifying phosphatidylcholine (PC) reductions and reciprocal lyso-phosphatidylcholine (LPC) increments. The values were expressed as PC/LPC ratios, with falling ratios indicating increasing PLA2 activity. These experiments were repeated 6 times on different occasions.
Fe-Mediated Lipid Peroxidation Experiments. Vesicles that had been formed with the 0, 5, 10, and 20% SM additions were incubated at 37°C with 50 µM FeHQ. The extent of lipid peroxidation was assessed after completing 30-min incubations by determining malondialdehyde (MDA) content (28). The results were compared with those observed in vesicles that received no FeHQ treatment.
Partial SM Depletion Experiments: Effects on PLA2-Mediated Phospholipid Degradation. In these experiments, vesicles were reconstituted without exogenous SM. Rather, they were coincubated with SMase (0.25 U/ml) to partially deplete SM. The impact of the SMase-mediated SM reductions (approximately 40%) on PLA2 (0.05 U/ml)-mediated PC degradation was assessed (coincubations ± one or both of the enzymes; n = 6 experiments assessing each treatment).
Effects of Membrane Fluidity on Fe- and PLA2-Mediated Damage. SM, like cholesterol, decreases membrane fluidity. To assess the impact of the latter on the expression of membrane damage, vesicles were treated with a membrane-fluidizing agent, and then its impacts on PLA2-mediated PC degradation and Fe-mediated lipid peroxidation were addressed, as follows.
PLA2 Activity. Vesicles were incubated with PLA2 as noted above either in the presence or absence of the membrane-fluidizing reagent A2C [2-(2-methoxyethoxy)ethyl 8-(cis-2octyl-cyclopropyl) octanoate; M-9010; Sigma Chemical Co.) (24). The A2C was solubilized in DMSO, added to achieve a final 0.5 mM A2C concentration. After completing 30-min incubations, PLA2 activity was assessed by determining PC/LPC ratios, as noted above. Non-PLA2-treated vesicles that were coincubated with and without A2C/A2C vehicle provided control PC/LPC values (n = 6 determinations each).
Fe-Mediated Lipid Peroxidation. The above experiment was repeated 4 times, substituting 50 µM FeHQ for the PLA2 challenge. At the completion of 30-min incubations, the extent of Fe-mediated lipid peroxidation was assessed by determining MDA concentrations, as noted above.
SMase Effects on HK-2 Cell Proliferation
Basal Conditions. HK-2 cells were seeded at low density (0.75
x 105 cells/well) for 24 h in 24-well cluster plates. They
were then exposed to either 0, 0.1, 0.25, 0.5, or 1.0 U/ml SMase for an
additional 48 h. At the completion of these incubations, cell
proliferation/cell number was gauged by a tetrazolium dye uptake assay
(methylthiotetrazole [MTT]), as described previously
(21). These experiments were
repeated 4 times on two separate occasions.
As a second indicator of SMase effects on HK-2 cell proliferation, two additional 24-well plates, prepared as noted above, were incubated for 48 h with 0, 0.25, or 0.5 U/ml SMase. Cell outgrowth was assessed by the crystal violet staining technique (29), as described previously (30).
Cell Proliferation after Fe-Mediated Injury
The following experiment assessed the impact of SMase on cell proliferation
of sublethally damaged HK-2 cells. To this end, eight 24-well plates were
cultured for 8 h and then exposed to a sublethal FeHQ dose (2.5 µM FeHQ for
18 h). After this treatment, the cells were cultured for an additional 48 h
with 0.5 U/ml SMase. Cocultured cells subjected to either continuous control
culture conditions, 48-h treatment with SMase but without a prior FeHQ
challenge, or the FeHQ challenge without subsequent SMase treatment were used
for comparison. After completing these protocols, relative cell numbers were
assessed by MTT assay.
Statistical Analyses
All values are presented as means ± 1 SEM. Statistical comparisons
were made by either paired or unpaired t test. If multiple
comparisons were made, the Bonferroni method was applied.
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Effects of SMase on Fe-Mediated Cell Injury
As shown in Figure 2, the
FeHQ challenge caused approximately 15% LDH release. The extent of this
cytotoxicity was increased two- to threefold when the Fe challenge was
conducted in the presence of SMase (Figure
2). Once again, SMase exerted no independent cytolytic effect.
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Effects of SMase Treatment on the Expression of Arachidonic Acid
Cytotoxicity
Incubating HK-2 cells with exogenous arachidonic acid (C20:4) caused
approximately 65% LDH release (Figure
3). Unlike the results obtained with the CAD and FeHQ challenges,
SMase decreased, rather than increased, this cytotoxicity.
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SMase Effects on HK-2 Cell Phospholipid, Cholesterol, and Ceramide
Content
Phospholipids. Incubating HK-2 cells for 14 h with SMase caused a
36% decline in absolute SM content (from 22.3 ± 2.2 to 14.2 ±
2.9 nmol/sample; P < 0.005). This was reflected by a relative
decrease in its overall contribution to total plasma membrane phospholipid
composition (from 11.7 to 8%) (Table
1). The percent decrement in SM was, in large part, matched by a
reciprocal increase in percentage of PC in the SMase-treated cells.
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Cholesterol. Exposing the HK-2 cells to 1 U/ml SMase for 14 h induced no change in their total cholesterol content (controls, 0.257 ± 0.03; SMase treatment, 0.259 ± 0.02; nmol cholesterol/nmol phospholipid phosphate; NS).
Ceramide. HPLC/MS analysis demonstrated C16 and C24, but not C22 ceramides (15) within HK-2 cells (Table 2). Varying degrees of fatty acid unsaturation were apparent within both the C16 and the C24 ceramide pools. Treatment with exogenous SMase caused surprisingly modest total ceramide increments (approximately 35 to 50% greater than that observed in control cells). The bulk of this increase was due to increases in the C16:0, C16:1, and C24:1 pools.
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Effect of Exogenous SM Treatment on HK-2 Cell Injury
As shown in Figure 4,
incubating HK-2 cells with exogenous SM alone did not affect cell viability.
The CAD challenge induced approximately 66% cell death. Approximately
two-thirds of this injury was eliminated by the SM treatment
(Figure 4, left). SM also
attenuated FeHQ-mediated cell injury (P < 0.0005), albeit to a
lesser degree (Figure 4 right).
(Note: The greater cell injury induced by the CAD and FeHQ challenges
in these experiments versus the SMase addition experiments reflects
the difference in time the cells were in culture [8 to 9 h versus 24
to 48 h] before applying the cytotoxic challenges. In general, HK-2 cells are
more vulnerable to injury if challenged 8 h versus 24 to 48 h after
establishing the cultures. The shorter time frame was used in the SM addition
experiments to increase cell injury, thereby allowing a more accurate
quantitative assessment of SM's cytoprotective effect.)
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Isolated Vesicle Experiments
Impact of SM Additions on PLA2-Mediated PC Degradation.
Control vesicles had no discernible LPC present (and hence, no PC/LPC ratios
are depicted in Figure 5). The
PLA2 challenge reduced vesicle PC content by 50 ± 4%
(P < 0.001). A reciprocal increase in LPC resulted, producing a
PC/LPC ratio of approximately 1.0 (Figure
5A). Raising vesicle SM content by exogenous SM addition caused a
significant blunting of PC degradation, as reflected by a significant
preservation of the PC/LPC ratio (Figure
5A) (*P < 0.05) during PLA2
treatment. This result was not dose-dependent, because essentially identical
ratios were obtained with 5, 10, or 20% SM addition.
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Impact of SM Depletion on PLA2-Mediated PC Degradation. SMase treatment of the vesicles decreased their SM content by 40 ± 4% (P < 0.001). There was no associated change in PC, and hence, no LPC was apparent. When these partially SM-depleted vesicles were challenged with PLA2, increased PC degradation/LPC generation resulted. This was reflected by a further lowering of the PC/LPC ratio, compared to that seen in vesicles subjected to PLA2 treatment without SMase (Figure 5B).
Impact of SM Additions on Fe-Mediated Lipid Peroxidation. Baseline vesicle MDA concentrations were <1.0 µM. This result was unaffected by SM additions. Fe addition raised control vesicle MDA concentrations to 6.9 ± 1.1 µM. This result was significantly reduced by prior incorporation of SM into the vesicles (6.4 ± 0.9, 6.1 ± 1.1, and 5.7 ± 1.1 µM; with 5, 10, and 20% SM supplementation; P < 0.03 versus non-SM-supplemented vesicles).
Effects of Membrane Fluidity on Fe- and PLA2-Mediated Damage. A2C had no independent effect on vesicle PC content, and no LPC was observed. However, A2C increased PLA2-mediated PC deacylation, lowering the PC/LPC ratio by an additional 20% (Figure 6A).
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When only A2C was added to the vesicles, no change in MDA concentrations resulted (Figure 6B). However, A2C approximately doubled MDA generation in the presence of the Fe challenge (Figure 6B).
SMase Effects on HK-2 Cell Proliferation
Impact on Normal HK-2 Cells. When otherwise normal HK-2 cells were
exposed to SMase treatment, a 10% suppression of HK-2 cell outgrowth was
noted, as assessed by the MTT assay. This effect was maximal at the 1 U/ml
SMase concentration (controls: 0.31 ± 0.01 MTT absorbance units; SMase
at 0.1, 0.25, 0.5, and 1.0 U/ml: 0.31 ± 0.01, 0.31 ± 0.01, 0.30
± 0.005, and 0.29 ± 0.01 absorbance units, respectively). These
results were statistically significant at and above the 0.50 U/ml
concentration (P < 0.05 to <0.0003). The crystal violet assay
confirmed this approximate 10% decrease in HK-2 outgrowth in the presence of
SMase (controls: 0.57 ± 0.02 absorbance units; 0.25 and 0.5 U/ml SMase:
0.48 ± 0.0.2 and 0.51 ± 0.02, respectively; P 
0.025 with each concentration).
Impact on Cell Proliferation after Sublethal Fe-Mediated Injury. The sublethal Fe challenge, by itself, caused a 10 ± 1% decrease in cell outgrowth (MTT assay) compared with cocultured control cells (P < 0.001). SMase addition further decreased cell outgrowth in these post-Fe-damaged cells (to 79 ± 2% of controls; P < 0.001). However, because SMase alone caused a 10 ± 1% decrease in MTT uptake/cell proliferation (P < 0.01), the 21% decrease in cell outgrowth in the Fe + SMase-treated cells could be attributed to additive, rather than synergistic, Fe + SMase growth inhibitory effects.
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To evaluate the impact of SMase activity on evolving tubular injury, HK-2 cells were exposed to exogenous SMase under basal conditions and during superimposed ATP depletion/Ca2+ overload or iron-induced oxidant attack. That SM resides predominantly in the outer plasma membrane leaflet assures that exogenous SMase gains ready SM access. The use of SMase also assures that a full array of physiologic ceramides (i.e., with differing constituent fatty acids) is generated directly within the plasma membrane. It is noteworthy that most physiologic ceramides are not commercially available, and those that are undergo minimal cell uptake (33). In the present studies, SMase-induced approximately 33% SM hydrolysis, decreasing it from approximately 12% to approximately 8% of the total cellular phospholipid mass. Despite this large SM reduction, only modest ceramide increments resulted. It should be recalled that with a normal 30:1 SM:ceramide ratio in HK-2 cells, 33% SM hydrolysis should yield an approximate 1000% ceramide increase. That total ceramide increased only by 33 to 50% indicates that the vast majority of generated ceramide was rapidly catabolized (presumably by ceramidases), thereby precluding marked ceramide increments. This finding of comparatively modest ceramide accumulation in the face of massive SM hydrolysis underscores our previous conclusion that the ceramide increments after acute tubular injury must stem, at least in part, from decreased ceramide catabolism, and not simply SM breakdown (13).
The present study demonstrates that SMase can substantially impact acute tubular cell damage. When HK-2 cells were challenged with ATP depletion/Ca2+ overload in the presence of SMase, an approximate doubling of LDH release resulted. SMase also exacerbated Fe-mediated cytotoxic death. These results cannot simply be explained by nonspecific SMase toxicity because SMase, by itself, caused no cell death. Furthermore, SMase attenuated, rather than exacerbated, arachidonic acid cytotoxicity, indicating the lack of a nonspecific "injury-provoking" effect. In sum, then, the present results indicate that SMase can differentially alter the evolution of acute tubular cell injury/cell death.
It is noteworthy that although SMase treatment generated only modest ceramide increments, it partially recapitulated results obtained with high (µM) dose exogenous ceramide additions: Both SMase and exogenous ceramide worsen ATP depletion/Ca2+ ionophore-induced injury, whereas each decrease arachidonic acid-mediated tubular cell death (13,15,16,17) (present data). In contrast, SMase exacerbated Fe-mediated cytotoxicity (present data), whereas exogenous ceramide produced the opposite result (13). This latter difference illustrates that SMase's impact on evolving cell injury is not explained simply by ceramide generation. Rather, SMase-mediated SM decrements are likely involved, as discussed below.
A previous study from this laboratory indicates that plasma membrane cholesterol is a critical determinant of cellular resistance to injury (24). This conclusion stems from observations that decreasing membrane cholesterol content (by decreasing synthesis or cell extraction) or its enzymatic modification (with cholesterol esterase or oxidase) dramatically increases tubular cell vulnerability to ATP depletion/Ca2+ ionophore or Fe-induced attack. A membrane-fluidizing reagent (A2C) recapitulated this injury potentiation (24), suggesting that cholesterol's influence on cell injury is mediated via its "anti-fluidity" effect. It is noteworthy that SM is tightly complexed to cholesterol within the plasma membrane, forming SM:cholesterol microdomains (1,2,3,4,31). Because SMase-mediated SM reductions undoubtedly disturb these domains, it might then abrogate cholesterol's cytoprotective effect. Alternatively, if SM, like cholesterol, has a direct cytoprotective action, SMase could promote injury directly by reducing membrane SM content.
To explore this latter possibility, SM's impact on cell injury was assessed by incubating HK-2 cells with exogenous SM for 8 h (allowing for membrane uptake), and then cellular susceptibility to ATP depletion/Ca2+ ionophore and Fe toxicity were assessed. In both instances, substantial decrements in lethal cell injury resulted. It is noteworthy that in pilot studies conducted without this 8-h pretreatment period, SM failed to confer this same protective action. Thus, the SM-mediated attenuation of cell injury cannot simply be explained by inhibition of cellular toxin uptake. Because SMase may, under certain circumstances, decrease cellular cholesterol content (4,25), it seemed possible that SMase's ability to exacerbate HK-2 cell injury could stem from reduction in cholesterol, rather than in SM. However, the fact that SMase did not alter HK-2 cell cholesterol content excludes this hypothesis. Finally, that SM supplementation of isolated vesicles directly blunted lipid peroxidation and PLA2-mediated PC deacylation provides additional strong support for the concept that SM can, indeed, confer direct cytoprotection by stabilizing plasma membranes against attack. That A2C exerted exactly the opposite effect as SM on PLA2- and Fe-mediated vesicle damage strongly suggests that SM's membrane protective action, as with cholesterol, arises from an anti-fluidity effect.
It is conceivable that SM's ability to confer membrane, and hence cellular, resistance to injury could extend beyond the specific issue of SMase-induced SM hydrolysis. First, during acute tubular cell injury, SM moves from its concentrated position within the brush-border membrane to the basolateral membrane, a consequence of cytoskeletal disruption/loss of cell polarity (18) ± SMase activity. Given that brush-border damage is the earliest and most prominent morphologic consequence of ischemic cell injury (34), it is tempting to postulate that these early SM losses contribute to this damage. Second, within 24 h of myohemoglobinuric renal injury, SM increments develop within renal cortex (26). It is noteworthy that this coincides with the emergence of renal tubular resistance to superimposed attack (the so-called state of "acquired cytoresistance") (35). While multiple mechanisms contribute to the latter (e.g., increased heme oxygenase, ferritin, cholesterol, heat shock proteins, etc.) (36,37,38), an increase in membrane SM content might also play a role. These two considerations underscore potential broad-based implications of altered SM expression on cellular resistance to superimposed attack.
In addition to altering tubular cell injury, SMase activity could potentially affect the evolution of ARF by retarding tubular cell proliferation after an ischemic or toxic insult. Noteworthy in this regard are reports that SMase can be antimitogenic, an effect linked to ceramide generation (10,11). To our knowledge, this issue has not been addressed previously in proximal tubular epithelia. The present experiments indicate that SMase can, indeed, inhibit HK-2 cell outgrowth, but the effect appears quite mild (approximately 10% reductions), as gauged by two independent cell proliferation assays. It is tempting to postulate that this negative effect would be magnified in sublethally damaged tubular cells that already have a diminished proliferative capacity. However, this does not appear to be the case, given that SMase's antiproliferative activity was essentially identical in control cells and in cells that were sublethally damaged by prior Fe-mediated injury.
In conclusion, the present study demonstrates that SMase activity can potentially exacerbate both ATP/Ca2+ overload and Fe-mediated oxidant cell death. This action does not simply reflect nonspecific SMase "toxicity," because the dose of SMase used, by itself, did not impact cell viability, and it decreased, rather than increased, arachidonic acid's cytotoxic effect. The ability of SMase to increase cell injury may stem from SM decrements, and not simply cytotoxic ceramide gains. This is based on observations that SM exerts membrane-protective effects, both in intact HK-2 cells as well as in isolated vesicles. Despite SMase's ability to evoke marked SM hydrolysis, surprisingly modest ceramide increments result. This suggests that high endogenous ceramidase activity within cells can markedly blunt ceramide accumulation, underscoring our previous assertion that the dramatic ceramide increments that develop during renal injury arise, in part, from ceramidase inhibition, and not simply SM hydrolysis. Finally, despite the fact that SMase can exert potent antiproliferative effects, its impact on proximal tubule cell growth appears quite modest, both under basal conditions and in the setting of sublethal tubular damage. If these results are relevant to in vivo renal injury, they suggest a greater potential for SMase to impact the induction, rather than the recovery, phase of ischemic and toxic ARF.
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Acknowledgments |
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Footnotes |
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References |
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F. Gueler, S. Rong, J.-K. Park, A. Fiebeler, J. Menne, M. Elger, D. N. Mueller, F. Hampich, R. Dechend, U. Kunter, et al. Postischemic Acute Renal Failure Is Reduced by Short-Term Statin Treatment in a Rat Model J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2288 - 2298. [Abstract] [Full Text] [PDF]
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 R. A. Zager and T. F. Kalhorn Changes in Free and Esterified Cholesterol : Hallmarks of Acute Renal Tubular Injury and Acquired Cytoresistance Am. J. Pathol., September 1, 2000; 157(3): 1007 - 1016. [Abstract] [Full Text] [PDF]
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